Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein

The TcTLE peptide (TLEEFSAKL) is a CD8⁺ T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8⁺ T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8⁺ T cells in HLA-A2⁺ chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8⁺ T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

The peptide-binding specificity of HLA-A*3001 demonstrates membership of the HLA-A3 supertype

Human leukocyte antigen class I (HLA-I) molecules are highly polymorphic peptide receptors, which select and present endogenously derived peptide epitopes to CD8+ cytotoxic T cells (CTL). The specificity of the HLA-I system is an important component of the overall specificity of the CTL immune system. Unfortunately, the large and rapidly increasing number of known HLA-I molecules seriously complicates a comprehensive analysis of the specificities of the entire HLA-I system (as of June 2008, the international HLA registry holds >1,650 unique HLA-I protein entries). In an attempt to reduce this complexity, it has been suggested to cluster the different HLA-I molecules into “supertypes” of largely overlapping peptide-binding specificities. Obviously, the HLA supertype concept is only valuable if membership can be assigned with reasonable accuracy. The supertype assignment of HLA-A*3001, a common HLA haplotype in populations of African descent, has variously been assigned to the A1, A3, or A24 supertypes. Using a biochemical HLA-A*3001 binding assay, and a large panel of nonamer peptides and peptide libraries, we here demonstrate that the specificity of HLA-A*3001 most closely resembles that of the HLA-A3 supertype. We discuss approaches to supertype assignment and underscore the importance of experimental verification.     

An integrated approach to epitope analysis I: Dimensional reduction, visualization and prediction of MHC binding using amino acid principal components and regression approaches

Background

Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8⁺ T cells from seropositive but not seronegative persons.

Results

Herein, when these peptides were administered with the universal CD4⁺T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam₂Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam₂Cys is an effective way to elicit IFN-γ producing CD8⁺ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1_224-232); AMLTAFFLR (GRA6_164-172); RSFKDLLKK (GRA7_134-142); STFWPCLLR (SAG2C_13-21); SSAYVFSVK(_SPA250-258); and AVVSLLRLLK(SPA_89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera.

Conclusions

Toxoplasma gondii peptides elicit HLA-A03 restricted, IFN-γ producing, CD8⁺ T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles.     

Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: biological validation of the proposed HLA A24 supertype

Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.     

Cross-Allele Cytotoxic T Lymphocyte Responses against 2009 Pandemic H1N1 Influenza A Virus among HLA-A24 and HLA-A3 Supertype-Positive Individuals

Lack of a universal vaccine against all serotypes of influenza A viruses and recent progress on T cell-related vaccines against influenza A virus illuminate the important role of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) in anti-influenza virus immunity. However, the diverse HLA alleles among humans complicate virus-specific cellular immunity research, and elucidation of cross-HLA allele T cell responses to influenza virus specificity requires further detailed work. An ideal CTL epitope-based vaccine would cover a broad spectrum of epitope antigens presented by most, if not all, of the HLAs. Here, we evaluated the 2009 pandemic influenza A (H1N1) virus-specific T cell responses among the HLA-A24⁺ population using a rationally designed peptide pool during the 2009 pandemic. Unexpectedly, cross-HLA allele T cell responses against the influenza A virus peptides were detected among both HLA-A11⁺ and HLA-A24⁺ donors. Furthermore, we found cross-responses in the entire HLA-A3 supertype population (including HLA-A11, -A31, -A33, and -A30). The cross-allele antigenic peptides within the peptide pool were identified and characterized, and the crystal structures of the major histocompatibility complex (MHC)-peptide complexes were determined. The subsequent HLA-A24-defined cross-allele peptides recognized by the HLA-A11⁺ population were shown to mildly bind to the HLA-A*1101 molecule. Together with the structural models, these results partially explain the cross-allele responses. Our findings elucidate the promiscuity of the cross-allele T cell responses against influenza A viruses and are beneficial for the development of a T cell epitope-based vaccine applied in a broader population.     

Identification of SART3-derived peptides having the potential to induce cancer-reactive cytotoxic T lymphocytes from prostate cancer patients with HLA-A3 supertype alleles

SART3-derived peptides applicable to prostate cancer patients with HLA-A3 supertype alleles were identified in order to expand the possibility of an anti-cancer vaccine, because the peptide vaccine candidates receiving the most attention thus far have been the HLA-A2 and HLA-A24 alleles. Twenty-nine SART3-derived peptides that were prepared based on the binding motif to the HLA-A3 supertype alleles (HLA-A11, -A31, and -A33) were first screened for their recognizability by immunoglobulin G (IgG) of prostate cancer patients and subsequently for the potential to induce peptide-specific cytotoxic T lymphocytes (CTLs) from HLA-A3 supertype⁺ prostate cancer patients. As a result, five SART3 peptides were frequently recognized by IgG, and two of them—SART3 _511–519 and SART3 _734–742—efficiently induced peptide-specific and cancer-reactive CTLs. Their cytotoxicity toward prostate cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results indicate that these two SART3 peptides could be promising candidates for peptide-based immunotherapy for HLA-A3 supertype⁺ prostate cancer patients. Grant sponsor This study was supported in part by KAKENHI (Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan) (no. 12213134 to K. Itoh, and no. 18591449 to M. Harada), Research Center of Innovative Cancer Therapy of 21st Century COE Program for Medical Science to K. Itoh, and the Ministry of Health, Labor and Welfare, Japan (15–17 to M. Harada).     

Functional analysis of frequently expressed Chinese rhesus macaque MHC class I molecules Mamu-A1*02601 and Mamu-B*08301 reveals HLA-A2 and HLA-A3 supertypic specificities

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques?? major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC?Cpeptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.     

Identification of Special AT-Rich Sequence Binding Protein 1 as a Novel Tumor Antigen Recognized by CD8+ T Cells: Implication for Cancer Immunotherapy

Background

A large number of human tumor-associated antigens that are recognized by CD8⁺ T cells in a human leukocyte antigen class I (HLA-I)-restricted fashion have been identified. Special AT-rich sequence binding protein 1 (SATB1) is highly expressed in many types of human cancers as part of their neoplastic phenotype, and up-regulation of SATB1 expression is essential for tumor survival and metastasis, thus this protein may serve as a rational target for cancer vaccines.

Methodology/Principal Findings

Twelve SATB1-derived peptides were predicted by an immuno-informatics approach based on the HLA-A*02 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from HLA-A*02⁺ healthy donors and/or HLA-A*02⁺ cancer patients. The recognition of HLA-A*02⁺ SATB1-expressing cancer cells was also tested. Among the twelve SATB1-derived peptides, SATB1_565–574 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and cancer patients. Importantly, SATB1_565–574-specific T cells recognized and killed HLA-A*02⁺ SATB1⁺ cancer cells in an HLA-I-restricted manner.

Conclusions/Significance

We have identified a novel HLA-A*02-restricted SATB1-derived peptide epitope recognized by CD8⁺ T cells, which, in turn, recognizes and kills HLA-A*02⁺ SATB1⁺ tumor cells. The SATB1-derived epitope identified may be used as a diagnostic marker as well as an immune target for development of cancer vaccines.     

Specific CD8+ T cell responses to HLA-A2 restricted MAGE-A3 p271–279 peptide in hepatocellular carcinoma patients without vaccination

The MAGE-A3 protein, one of the promising tumor antigens for immunotherapy, is highly expressed in human hepatocellular carcinoma (HCC). In this study, we estimated the specific CD8⁺ T cell immune response to MAGE-A3 p271–279 peptide (M3₂₇₁) in the peripheral blood of HCC patients without antigen vaccination in order to evaluate its immunotherapeutic potential in these patients. After expansion in vitro, the functional IFN-γ producing M3₂₇₁ specific CD8⁺ T cells were detected in 30.8% (8/26) of HLA-A2⁺MAGE-A3⁺ HCC patients. The effector CD8⁺ T cells could release cytotoxic molecules of granzyme B and perforin after restimulation with natural HLA-A2⁺MAGE-A3⁺ HCC cell lines in the samples tested. The functional supertype of HLA-A2 in the presentation of HLA-A*0201 restricted M3₂₇₁ peptide has been identified in the Chinese HCC patients of Han ethnicity, that widely expanded the applicability of this tumor peptide vaccine in Chinese HCC patients. Thus, the functionally detectable pre-existence of M3₂₇₁-specific CD8⁺ T cells in HCC patients makes M3₂₇₁ a potential target for immunotherapy in these patients. The responsive CD8⁺ T cells to both NY-ESO-1 and MAGE-A3 antigens provide a rationale for the application of a bivalent vaccine in HCC patients with tumors expressing both antigens. H-G Zhang, H-S Chen, and J-R Peng are contributed equally to this paper.     

HLA-A2 and B35 Restricted Hantaan Virus Nucleoprotein CD8+ T-Cell Epitope-Specific Immune Response Correlates with Milder Disease in Hemorrhagic Fever with Renal Syndrome

Background

Hantaan virus (HTNV) infection in humans is a serious public health concern in Asia. A potent T cell activation peptide vaccine from HTNV structure protein represents a promising immunotherapy for disease control. However, the T cell epitopes of the HTNV restricted by the HLA alleles and the role of epitope-specific T cell response after HTNV infection remain largely unexplored.

Methodology/Principal Findings

Five well-conserved novel CD8⁺ T-cell epitopes of the HTNV nucleoprotein restricted by the most popular HLA alleles in Chinese Han population were defined with interferon-γ enzyme-linked immunospot assay in 37 patients infected with HTNV during hospitalization. Two epitopes aa129–aa137 and aa131–aa139 restricted by HLA-A2 and B35, respectively, were selected to evaluate the epitope-specific CD8⁺ T-cell response. HLA-peptide pentamer complex staining showed that the frequency of single epitope-specific CD8⁺ T cell could be detected in patients (95% confidence interval for aa129–aa137: 0.080%–0.208%; for aa131–aa139: 0.030%–0.094%). The frequency of epitope-specific pentamer⁺ CD8⁺ T-cell response was much higher in mild/moderate patients than in severe/critical ones at the acute stage of the disease. Moreover, the frequency of epitope-specific CD8⁺ T cells at acute stage was inversely associated with the peak level of serum creatinine and was positively associated with the nadir platelet counts during the hospitalization. The intracellular cytokine staining and the proliferation assay showed that the effective epitope-specific CD8⁺ T cells were characterized with the production of interferon-γ, expression of CD69 and the strong capacity of proliferation.

Conclusion/Significance

The novel HLA class I restricted HTNV nucleoprotein epitopes-specific CD8⁺ T-cell responses would be closely related with the progression and the severity of the disease, which could provide the first step toward effective peptide vaccine development against HTNV infection in humans.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

HLA Class I-T cell epitopes from trans-sialidase proteins reveal functionally distinct subsets of CD8+ T cells in chronic Chagas disease

Background

Previously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8⁺ T cell responses in HLA-A0201⁺ individuals chronically infected by T. cruzi.

Methods and Findings

Herein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8⁺ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8⁺ T cell responses to promiscuous epitopes revealed that the CD8⁺ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-γ alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-γ/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection.

Conclusions

This study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease.     

HLA Allele E*01:01 Is Associated with a Reduced Risk of EBV-Related Classical Hodgkin Lymphoma Independently of HLA-A*01/*02

Background

An inefficient immune response against Epstein-Barr virus (EBV) infection is related to the pathogenesis of a subgroup of classical Hodgkin lymphomas (cHL). Some EBV immune-evasion mechanisms target HLA presentation, including the non-classical HLA-E molecule. HLA-E can be recognized by T cells via the TCR, and it also regulates natural killer (NK) cell signaling through the inhibitory CD94/NKG2A receptor. Some evidences indicate that EBV-infected B-cells promote the proliferation of NK subsets bearing CD94/NKG2A, suggesting a relevant function of these cells in EBV control. Variations in CD94/NKG2A-HLA-E interactions could affect NK cell-mediated immunity and, consequently, play a role in EBV-driven transformation and lymphomagenesis. The two most common HLA-E alleles, E*01:01 and E*01:03, differ by a single amino acid change that modifies the molecule function. We hypothesized that the functional differences in these variants might participate in the pathogenicity of EBV.

Aim

We studied two series of cHL patients, both with EBV-positive and-negative cases, and a cohort of unrelated controls, to assess the impact of HLA-E variants on EBV-related cHL susceptibility.

Results

We found that the genotypes with at least one copy of E*01:01 (i.e., E*01:01 homozygous and heterozygous) were underrepresented among cHL patients from both series compared to controls (72.6% and 71.6% vs 83%, p = 0.001). After stratification by EBV status, we found low rates of E*01:01-carriers mainly among EBV-positive cases (67.6%). These reduced frequencies are seen independently of other factors such as age, gender, HLA-A*01 and HLA-A*02, HLA alleles positively and negatively associated with the disease (adjusted OR = 0.4, p = 0.001). Furthermore, alleles from both HLA loci exert a cumulative effect on EBV-associated cHL susceptibility.

Conclusions

These results indicate that E*01:01 is a novel protective genetic factor in EBV-associated cHL and support a role for HLA-E recognition on the control of EBV infection and lymphomagenesis.     

Classification of A1- and A24-supertype molecules by analysis of their MHC-peptide binding repertoires

At the functional level, the majority of human leukocyte antigen (HLA) class I MHC variants can be classified into about ten different major groups, or supertypes, characterized by overlapping peptide binding motifs and repertoires. Previous studies have detailed the peptide binding specificity of the HLA A2, A3, B7, and B44 supertypes, and predicted, on the basis of MHC pocket structures, known motifs, or the sequence of T cell epitopes, the existence of the HLA A1 and A24 supertypes. Direct experimental validation of the A1 and A24 supertypes, however, has been lacking. In the current study, the peptide-binding repertoires and main anchor specificities of several common HLA A molecules (A*0101, A*2301, A*2402, A*2601, A*2902, and A*3002) predicted to be members of the A1 or A24 supertypes were analyzed and defined using single amino acid substituted peptides and a large peptide library. Based on the present findings, the A1 supertype includes A*0101, A*2601, A*2902, and A*3002, whereas the A24 supertype includes A*2301 and A*2402. Interestingly, A*2902 is associated with a motif and peptide binding repertoire that overlaps significantly with those of all of the A1- and A24-supertype molecules studied, representing—to our knowledge—the first report of significant cross-reactivity among molecules belonging to different supertypes.     

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8⁺ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8⁺ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8⁺ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8⁺ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8⁺ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2_320-331 and A11/PB2_323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M_41-49, A11/NS1_186-195 and A11/NP_511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8⁺ T cell epitopes has implications for understanding how CD8⁺ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.     

Improving MHC binding peptide prediction by incorporating binding data of auxiliary MHC molecules

MOTIVATION: Various computational methods have been proposed to tackle the problem of predicting the peptide binding ability for a specific MHC molecule. These methods are based on known binding peptide sequences. However, current available peptide databases do not have very abundant amounts of examples and are highly redundant. Existing studies show that MHC molecules can be classified into supertypes in terms of peptide-binding specificities. Therefore, we first give a method for reducing the redundancy in a given dataset based on information entropy, then present a novel approach for prediction by learning a predictive model from a dataset of binders for not only the molecule of interest but also for other MHC molecules. RESULTS: We experimented on the HLA-A family with the binding nonamers of A1 supertype (HLA-A*0101, A*2601, A*2902, A*3002), A2 supertype (A*0201, A*0202, A*0203, A*0206, A*6802), A3 supertype (A*0301, A*1101, A*3101, A*3301, A*6801) and A24 supertype (A*2301 and A*2402), whose data were collected from six publicly available peptide databases and two private sources. The results show that our approach significantly improves the prediction accuracy of peptides that bind a specific HLA molecule when we combine binding data of HLA molecules in the same supertype. Our approach can thus be used to help find new binders for MHC molecules.     

Survival and HLA-B*57 in HIV/HCV Co-Infected Patients on Highly Active Antiretroviral Therapy (HAART)

Background and aims

HLA class I alleles, in particular HLA-B*57, constitute the most consistent host factor determining outcomes in untreated HCV- and HIV-infection. In this prospective cohort study, we analysed the impact of HLA class I alleles on all-cause mortality in patients with HIV-, HCV- and HIV/HCV- co-infection receiving HAART.

Methods

In 2003 HLA-A and B alleles were determined and patients were prospectively followed in 3-month intervals until 2013 or death. HLA-A and B alleles were determined by strand-specific oligonucleotide hybridisation and PCR in 468 Caucasian patients with HCV- (n=120), HIV- (n=186) and HIV/HCV-infection (n=162). All patients with HIV-infection were on HAART. In each patient group, HLA class I-associated survival was analysed by Kaplan-Meier method and Cox regression analysis.

Results

At recruitment the proportion of patients carrying a HLA-B*57 allele differed between HIV- (12.9%) and HCV-infection (4.2%). Kaplan Meier analysis revealed significantly increased mortality in HLA-B*57-positive patients with HIV-infection (p=0.032) and HIV/HCV-co-infection (p=0.004), which was apparently linked to non-viral infections. Cox logistic regression analysis confirmed HLA-B*57 (p=0.001), serum gamma-glutamyltranspeptidase (p=0.003), serum bilirubin (p=0.022) and CD4 counts (p=0.041) as independent predictors of death in HIV-infected patients.

Conclusion

Differences in the prevalence of HLA-B*57 at study entry between HIV- and HCV- infected patients may reflect immune selection in the absence of antiviral therapy. When patients were treated with HAART, however, HLA-B*57 was associated with increased mortality and risk to die from bacterial infections and sepsis, suggesting an ambiguous role of HLA-B*57 for survival in HIV/HCV infection depending on the circumstances.     

UreB immunodominant epitope-specific CD8+ T-cell responses were beneficial in reducing gastric symptoms in Helicobacter pylori-infected individuals

Background and aims

Although Helicobacter pylori is recognized as an extracellular infection bacterium, it can lead to an increase in the number of CD8⁺ T cells after infection. At present, the characteristics of H. pylori antigen-specific CD8⁺ T cells and the epitope response have not been elucidated. This study was focused on putative protective antigen UreB to detect specific CD8⁺ T-cell responses in vitro and screen for predominant response epitopes.

Methods

The PBMCs collected from H. pylori-infected individuals were stimulated by UreB peptide pools in vitro to identify the immunodominant CD8⁺ T-cell epitopes. Furthermore, their HLA restriction characteristics were detected accordingly by NGS. Finally, the relationship between immunodominant responses and appearance of gastric symptoms after H. pylori infection was conducted.

Results

UreB-specific CD8⁺ T-cell responses were detected in H. pylori-infected individuals. Three of UreB dominant epitopes (A-2 (UreB_443–451: GVKPNMIIK), B-4 (UreB_420–428: SEYVGSVEV), and C-1 (UreB_5–13: SRKEYVSMY)) were firstly identified and mainly presented by HLA-A*1101, HLA-B*4001 and HLA-C*0702 alleles, respectively. C-1 responses were mostly occurred in H. pylori-infected subjects without gastric symptoms and may alleviate the degree of gastric inflammation.

Conclusions

The UreB dominant epitope-specific CD8⁺ T-cell response was closely related to the gastric symptoms after H. pylori infection, and the C-1 (UreB_5-13) dominant peptides may be protective epitopes.